The extracellular matrix (ECM), often referred to as connective tissue, is the complex structure that surrounds and supports cells in mammalian tissue. It is composed primarily of three classes of biomolecules: structural proteins (collagen and elastin), specialized proteins (e.g., laminin, fibronectin, fibrillin), and proteoglycans (core proteins linked to repeating disaccharides known as glycosaminoglycans or GAGs) (Pepper 2001). The ECM is vital for the maintenance and differentiation of many cell types, including the endothelium. In addition, it plays a role in the formation of new blood vessels from pre-existing ones, a process known as angiogenesis (Folkman 1995; Risau 1997).
Most normal cells cannot survive unless anchored to the ECM. This anchoring is mediated by heterodimeric transmembrane glycoproteins called integrins, which act as cellular adhesion receptors. Integrins are composed of non-covalently associated α and β subunits. Sixteen α subunits and eight β subunits have been identified, and over 20 different combinations of these subunits have been found. For example, integrin αvβ3 is a receptor on the surface of endothelial cells in growing blood vessels. It binds angiogenic endothelial cells, enabling them to form new blood vessels. Integrins anchor cells to their surroundings by mediating cell-matrix and cell-cell interactions. The extracellular portion of the integrin binds to collagen, laminin, or fibronectin, while the intracellular portion binds to actin filaments or the cytoskeleton. Extracellular binding to matrix proteins is dependent in large part on recognition of an RGD motif in the extracellular proteins. Fibronectin is the prototype RGD-containing protein.
Angiogenesis is the formation of new blood vessels from preexisting ones. Under normal conditions, angiogenesis is subject to tight physiological regulation, and the proliferation of endothelial cells is very low. Increased angiogenesis normally occurs in wound healing, embryonic development, and the monthly growth of the uterine lining in menstruating females. However, there are other situations in which increased angiogenesis is associated with a pathological condition. Uncontrolled angiogenesis has been associated with tumor growth, tumor metastasis, diabetic retinopathy, rheumatoid arthritis, and cardiovascular disease (Folkman 1995).
The endothelial cells that make up blood vessels generally remain in a quiescent state until they receive an angiogenic signal from their microenvironment. These signals may be triggered by wounds, inflammation, or disease. The angiogenic signal activates the endothelium and elicits a cascade of events that leads to new vessel formation: induction of proteases, degradation of the basement membrane, migration of endothelial cells into interstitial space, endothelial cell proliferation, lumen formation, generation of new basement membrane, fusion of new vessels, and initiation of blood flow.
The first step in the angiogenic cascade is the release of proteases such as matrix metalloproteinases (MMPs) by endothelial cells. These proteases degrade the basement membrane, a specialized type of ECM. The basement membrane is a storage depot for many angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Degradation of the basement membrane releases these angiogenic growth factors, which in turn propagates the angiogenic cascade. Degradation of the ECM also results in the release of ECM protein fragments, several of which have been implicated in the modulation of angiogenesis. Some of these fragments (e.g., an Mr 25,000 thrombospondin fragment) promote angiogenesis, while other fragments (e.g., endostatin derived from collagen XVIII, angiostatin derived from plasminogen, the noncollagenous domains of collagen IV, and several thrombospondin peptides) inhibit it (Taraboletti 2000; O'Reilly 1997; O'Reilly 1994; Maeshima 2001; Tolsma 1993).
Endothelial cells in blood vessels are in contact with a basement membrane that contains laminin, a large ubiquitous glycoprotein that exists in twelve different isoforms. Laminin is composed of three chains (α, β, and γ). Five different α, three β, and three γ chains have been identified. Ten of the twelve different heterotrimeric isoforms contain the γ1 chain (Timpl 1994; Burgeson 1994; Miner 1997). The identity of the laminin isoforms present in the endothelial cell matrix has not been determined. However, polyclonal antibodies to laminin-1 (composed of α1, β1, and γ1) recognize the matrix, suggesting the presence of at least one of these three chains. Laminin-1 promotes the attachment of endothelial cells in vivo, and the cells differentiate into capillary-like structures when plated on a laminin-1-rich basement membrane, such as Matrigel (Kubota 1988). Multiple binding sites for tumor cells have been identified on laminin-1 (Nomizu 1995; Nomizu 1997; Nomizu 1998).
More than twenty peptides from laminin-1 have been identified that can promote angiogenesis in vivo (Malinda 1999; Ponce 1999). These include eight peptides from the α1 chain, five from the β1 chain, and seven from the γ1 chain. Two of the most potent angiogenic peptide sites, A13 and C16, are redundant angiogenic sites present in homologous regions of the α1 and γ1 chains, respectively (Ponce 2003a; Kuratomi 2002; Kuratomi 1999). These peptide sequences bind to the endothelial cell integrins αvβ3 and α5β1, and have been shown to promote adhesion, tube formation, and angiogenesis in the chick chorioallantoic membrane (CAM) assay (Ponce 2001). The mechanism of action of these peptides has not yet been identified. Although they bind to integrins, they do not seem to signal through mitogen-activated protein kinase or several serine or threonine kinases. Eleven of the thirteen laminin proteins contain γ1 chains, meaning that the C16 sequence is present in eleven laminins (Colognato 2001). In addition, the A13 sequence is highly conserved in the laminin α chains. This means that several of the laminins, including laminin-1 and laminin-3, contain the A13 sequence twice (Nomizu 2001).
Because of the putative significance of the C16 sequence in angiogenesis and its related diseases, it has been important to identify antagonists capable of blocking its activity. One such antagonist is the scrambled peptide sequence C16S, which has been shown to inhibit C16 and bFGF-induced angiogenesis in the CAM assay (Ponce 2001). The methods disclosed in the present invention utilize a modified C16 peptide sequence, C16Y, which is at least five times more potent than C16S. C16Y inhibits choroidal neovascularization (CN) in vivo, in addition to inhibiting in vivo angiogenesis and tumor growth in mice (Ponce 2003b). Based on determination of its minimum active sequence, C16Y has been shown to share homology with fibronectin.
Unregulated angiogenesis is associated with the change of tumors from a quiescent state to a malignant state. Tumors require an extensive capillary network to grow and metastasize. Normally, a solid tumor will not grow beyond approximately 2 mm without the development of new blood vessels. Pathological or unregulated angiogenesis in the eye (ocular neovascularization) is the most common cause of blindness, and has been implicated in roughly twenty different eye diseases. The primary types of ocular neovascularization are retinal neovascularization, choroidal neovascularization, corneal neovascularization, and iris neovascularization.
Retinal neovascularization is the development of new blood vessels originating from the retinal veins and growing into the vitreous. Retinal neovascularization is associated with diabetic retinopathy, retinopathy of prematurity, central vein occlusion, and other retinal diseases. Diabetic retinopathy is responsible for 13-18% of newly reported cases of blindness (Kohner 1975), and it is the leading cause of legal blindness in people under 65 years old. Choroidal neovascularization (CN) is the development of new blood vessels in the vascular choroid, an area made up of large choroidal vessels and the choriocapillaris. The choriocapillaris is located next to the retinal pigmented epithelium and Bruch's membrane, and provides vascular support to the outer retina. CN is associated with a variety of diseases, including age-related macular degeneration (AMD) and high myopia. AMD is the leading cause of irreversible vision loss in world for people over 50 years old (Votruba 2001). Iris neovascularization, or rubeosis, often leads to the development of neovascular glaucoma. Corneal neovascularization, often associated with the use of contact lenses, can lead to vision loss.